The incidence of bacterial and fungal infections has increased dramatically over the last decade. One reason has been the increasing number of infections by human immunodeficiency virus (HIV), which causes AIDS.
AIDS is a vital infection targeting the CD4 lymphocytes of the immune system, leading to severe immunodeficiency. The World Health Organization estimates that at least 10 million people have been infected worldwide. J. M. Mann, J. Infect. Dis., 165:245-250 (1992). In the United States, over 1 million are believed to be infected and over 250,000 have developed AIDS. S. M. Schnittman et al., Advances in Internal Medicine, 39:305-355 (1994). A rapid spread of AIDS is predicted for southeast Asia and India, home to more than one third of the world's population.
The morbidity and mortality associated with AIDS result primarily from subsequent infections, often with fungal pathogens. Fungal infections affect various organs and tissues, including lungs, mucous membranes, brain, and lymph nodes. These infections weaken patients, resulting frequently in hospitalization and sometimes in death. Fungal infections cause about 20% of AIDS-related deaths.
AIDS-related fungal infections mainly involve Candida albicans, Cryptococcus neoformans, Aspergillus and Histoplasma, with Rhizopus, Mucor, and Blastococcus dermatitidis less frequent. Candidosis, resulting from C. albicans, comprises about 80% of fungal infections in AIDS patients. Oropharyngeal candidosis, common among AIDS patients, is rarely fatal but is quite painful. Holmberg et al., J. Infect. Dis., 18:179-184 (1986). Cryptococcosis, resulting from Cryptococcus neoformans, is less common but more likely to be lethal; it causes mortality of about 17% in its initial stages, 50 to 100% if relapse occurs. W. P. Powderly, Textbook of AIDS Medicine (S. Broder, T. C. Merigan Jr. & D. Bolgnesi, Eds.), William & Wilkins, Baltimore, Md. (1944), p. 345-357. Disseminated histoplasmosis is also quite lethal, with the chance of survival believed to be less than 60%.
In addition to AIDS patients, other populations are also immunocompromised and thus at great risk of infection. D. L. Brawner et al., Clinical Microbiol., 27:1335-1341 (1989). Patients undergoing organ and bone marrow transplants are deliberately given immunosuppressants to prevent transplant rejection. Immunosuppression is incidental in other treatments, including cancer chemotherapy, oropharyngeal irradiation, antimicrobial chemotherapy, and corticosteroid treatment. Diabetes and indwelling catheters can also predispose patients to opportunistic infections. Another consideration is an aging population, since immune functions generally decline with age. G. B. Sefano et al., "Aging and Cellular Defense Mechanisms," Ann. N.Y. Acad. Sci., 633:396 (1992).
Antibiotics have long been the first line of defense against infectious diseases. Many antibiotics with different modes of action are available, but few are antifungal. Moreover, the antifungal agents available are inadequate to treat many of the superficial and systemic mycoses that are prevalent.
The available antifungal agents comprise a small number of chemical types, such as polyene macrolides, synthetic azole compounds, griseofulvin, and 5-fluorocytosine. Especially active are polyene macrolides such as amphotericin B and nystatin. Introduced more than 30 years ago, amphotericin B is still the most potent antifungal drug for the treatment of deep-seated mycoses. Antifungal polyenes fight fungal pathogens by binding ergosterol, an essential sterol component of the fungal membrane. This binding allows formation of channels in the membrane and subsequent leakage of vital nutrients.
Polyenes may, however, cause severe side reactions. In addition to ergosterol, polyenes bind with lower affinity to cholesterol, a vital sterol in humans. As a result, polyenes may cause fever, disseminated intravascular coagulation, and even severe nephrotoxicity. Such nephrotoxicity may ultimately limit the dose administered, especially in conjunction with other nephrotoxic agents. J. Bratjburg et al., Antimicrob. Agents Chemother., 34:183-188 (1990). Polyenes must then be administered parenterally at low doses over long periods, necessitating hospitalization and prolonged intravenous access. So in spite of the proven worth of amphotericin B, there is reluctance to use it.
In addition to polyenes, a number of azole antifungal agents have been introduced into clinical practice. This group includes imidazoles (ketoconazole and econazole) and the more recently introduced triazoles (fluconazole and itraconazole). Unlike polyenes, azoles effect antifungal activity by inhibiting a fungal P450 methylase involved in sterol biosynthesis.
Like polyenes, azoles may also cause a number of undesired side reactions. Ketoconazole and econazole also inhibit the biosynthesis of human phospholipids and sterols, as well as the activity of human P450 enzymes, mitochondrial cytochrome oxidase, and membrane-bound enzymes (e.g., plasma membrane ATPase). In addition, they are rapidly eliminated from the body, necessitating multiple dosing.
The triazoles inhibit their target fungal demethylase with greater specificity than the imidazoles. They have been widely used to treat superficial mycoses and, more recently, systemic mycoses. Since its introduction in 1988, fluconazole has been used to treat more than 15 million patients, including over 250,000 AIDS patients (A. A. Hitchcock, Biochemical Society Transactions). It has become the agent of choice for systemic candidosis (associated with cancer and organ transplants), oropharyngeal candidosis (associated with AIDS), and cryptococcal meningitis (also associated with AIDS).
Unfortunately, fluconazole and all other currently available azole antifungal agents have a number of flaws. First, they are fungistatic; i.e., they suppress the growth of but do not kill the pathogen. Such fungistatic activity is a major flaw in treatment of immunocompromised patients, who may suffer relapses. Second, resistance may be emerging. L. Willocks et al., J. Antimicrob. Chemother., 28:937-9039 (1991). Although still relatively uncommon, resistant C. albicans strains have been reported after prolonged treatment with ketoconazole.
5-Fluorocytosine is a pyrimidine antifungal agent that is currently available. Originally developed as an antitumor agent, it acts by interfering with fungal DNA. After administration, it is transported into a susceptible fungal cell, deaminated to yield 5-fluorouracil, and subsequently converted to 5-fluorouridine triphosphate and 5-fluorodeoxyuridylate. The latter compound is a precursor of aberrant DNA, thus leading to aberrant protein. Although it has the advantage of oral availability, 5-fluorocytosine's usefulness is limited by the rapid emergence of resistance. In two studies cited by Kerridge et al., Drugs of Today 24:705-715 (1988), about 40% of strains examined were found to be partially resistant.
Griseofulvin is a natural product that affects the assembly of tubulin into microtubules (K. J. Weber et al., J. Mol. Biol., 102:817-829 (1977). It is orally active, but its use is limited to topical dermatophytic infections.
Allylamines (naftifine and terbinafine) have been introduced for the treatment of dermatophytosis. These compounds inhibit squalene epoxidase, an enzyme involved in sterol biosynthesis. Like several other antifungal agents currently available, the allylamines have limited spectra and, therefore, limited usefulness.
In short, we face an increasing risk of fungal infection, armed with a number of drugs having various drawbacks. A need exists for new antifungal agents and methods for identification thereof.
To find and develop new anti-fungal agents, a new method of looking for them may be required. As noted by one researcher, "The major problem in obtaining clinically active anti-fungal chemotherapies stems from the fact that, in fungal infections, we are dealing with a eukaryotic pathogen in a eukaryotic host and from a structural point of view the two do not greatly differ from each other. Thus specific targets for attack by chemotherapy are not as evident as in the case of bacterial pathogens." R. A. Calderone et al., Microbiol. Revs., 55:1-20 (1991).
One under-exploited target is the fungal cell wall. The cell wall is essential to growth of the yeast, because it contains many cell surface ligands and receptors that promote colonization of host cells and tissues. It is also essential to the protection of the yeast, because it provides rigidity to protect against osmotic injury. Since the fungal cell wall does not exist in man, it should provide differential screening targets--i.e., molecules identified should be more toxic to fungi than to man.
The synthesis of the cell wall in S. cerevisiae is mediated by a number of genes. The existence of temperature-sensitive mutations in S. cerevisiae causing cell lysis at non-permissive temperatures has been reported. L. H. Hartwell et al., Science, 183:46-51 (1974). The mutations were mapped to eight unlinked loci called CLY1-8. Hawthorne et al., Genetics 74:33-54 The lysis phenotype of these mutations suggests that these genes and their products can be utilized as differential targets in the search for antifungal agents.
S. cerevisiae has often been used as a surrogate for C. albicans in the development of screens for anti-fungal agents. D. R. Kirsch et al., Current Opinion in Biotechnology, 4:543-552 (1993). A vast amount of literature already exists about the physiology and genetics of S. cerevisiae and this organism is genetically more manipulable than is C. albicans, making it attractive as a test organism for study. Thus, genes involved in cell wall morphogenesis of S. cerevisiae may be used in screens for compounds that selectively inhibit yeasts and other fungi.